PUBLICATION

High-throughput target-selected gene inactivation in zebrafish

Authors
Kettleborough, R.N., Bruijn, E., Eeden, F., Cuppen, E., and Stemple, D.L.
ID
ZDB-PUB-110921-8
Date
2011
Source
Methods in cell biology   104: 121-127 (Chapter)
Registered Authors
Cuppen, Edwin, Kettleborough, Ross, Stemple, Derek L.
Keywords
zebrafish, mutagenesis, reverse genetics, TILLING, mutation detection, sequencing
MeSH Terms
  • Animals
  • Animals, Genetically Modified
  • DNA Mutational Analysis/methods*
  • Ethylnitrosourea/pharmacology
  • Exons
  • Female
  • Gene Library
  • Male
  • Mutagenesis
  • Mutagens/pharmacology
  • Polymerase Chain Reaction
  • Reverse Genetics/methods
  • Zebrafish/genetics*
PubMed
21924159 Full text @ Meth. Cell. Biol.
Abstract
There is an increasing requirement for efficient reverse genetics in the zebrafish, Here we describe a method that takes advantage of conventional mutagenized libraries (identical to ones used in forward screens) and re-sequencing to identify ENU-induced mutations in genes of interest. The efficiency of TILLING (Targeting Induced Local Legions IN Genomes) depends on the rate of mutagenesis in the library being screened, the amount of base pairs screened, and the ability to effectively identify and retrieve mutations on interest. Here we show that by improving the mutagenesis protocol, using in silico methods to predict codon changes for target selection, efficient PCR and re-sequencing, and accurate mutation detection we can vastly improve current TILLING protocols. Importantly it is also possible to use this method for screening for splice and mis-sense mutations, and with even a relatively small library, there is a high chance of identifying mutations across any given gene.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping